The goal of this research is to determine the molecular mechanisms by which insulin and nutrients control the synthesis of two key enzymes of energy metabolism. Fatty acid synthase (FAS) plays central role in de novo lipogenesis by catalyzing all the reactions involved in the conversion of acetyl CoA and malonyl CoA to palmitate. Phosphofructokinase (PFK-1) catalyzes the committed step in glycolysis. We have cloned cDNA sequences coding for FAS PFK and other insulin-inducible mRNAs. The level of the mRNAs are induced dramatically when insulin is given to diabetic animals or when a high carbohydrate diet is fed to previously starved mice. This research will further characterize how hormones and nutrients regulate expression of genes coding for these enzymes in cultured cells. Genomic DNA clones for FAS and PFK will be isolated and the overall structures of the transcription unit, intervening sequences and 5' flanking regions will be characterized. Fusion genes linking the putative regulatory sequences of FAS and PFK genes to reporter enzyme coding sequences will be introduced into an insulin-responsive cell line to elicit hormone dependent expression of these recombinants. This approach in combination with in vitro mutagenesis will be used to identify putative insulin-response element. Finally, the trans-acting factor which interacts specifically with the insulin regulatory sequences which mediate insulin action will be searched.